Herpes Simplex Virus-1 (HSV-1) remains a significant public health problem, causing blindness, encephalitis, and mucocutaneous ulcerative disease. It also contributes to major morbidity and mortality among immunosuppressed populations despite current efforts at vaccine and drug development. The long-term objective is to develop a novel approach to improve host defense against HSV-1. RNA interference (RNAi) is a gene suppression mechanism in which short fragments of double-stranded RNA, 21-23 bp, activate a RNA-inducible Suppression Complex (RISC). The activated RISC-siRNA binds to messenger RNA with sequence complementarity and bisects the targeted mRNA. This prevents translation into intact protein and facilitates mRNA degradation, effectively suppressing gene expression. RNAi emerged as a functional antiviral system in the plant kingdom. In mammals, RNAi does not appear to be a mechanism of immunity. However, the protein machinery of RNAi remains intact in mammalian cells, and we propose that this can be utilized against HSV-1. Our preliminary data with HSV-1 glycoprotein E (gE) shows that keratinocytes do express RNAi activity and that gE can be specifically suppressed (knockdown). The limitations of partial transfection efficiency and short duration of effect make RNAi poorly amenable as direct antiviral therapy. Therefore, we would like to develop and alternative approach to HSV treatment using a novel application of RNAi. We hypothesize that RNAi can be used to enhance HSV-specific immunity by suppressing viral immune evasion genes. Selected immune evasion properties from wild type virus of an infected host will be removed using exogenous siRNA, thereby producing immunologically unmasked infected cells. Aim 1 will test this hypothesis in vitro by determining whether a RNAi-mediated knockdown of gE prevents the evasion of antibody and complement by wild type virus. Aim 2 will test whether the knockdown of ICP47 is able to prevent the downregulation of major histocompatibility complex I and improve antigen presentation to T cells in vitro. Even a few such modified infected cells may be sufficient to enhance HSV-specific immunity in vivo. Aim 3 will focus on the development of a RNAi-mediated therapeutic vaccine in vivo. Together, the proposed studies will develop a RNAi-based immune enhancement model against HSV-1. [unreadable] [unreadable]